Abstract
An important breakthrough in the field of genome engineering was the discovery of the modular T ranscription A ctivator- L ike E ffector (TALE) DNA binding domain and the development of TALE n ucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species. Underlying the boom in genome engineering is the striking simplicity and low cost of engineering new specificities of TALENs and other sequence-specific nucleases. In this chapter, we describe a rapid, inexpensive, and user-friendly protocol for custom TALEN construction based on one of the most popular TALEN assembly platforms, the Golden Gate cloning method. Using this protocol, ready-to-use TALENs with specificity for targets 13-32 bp long are constructed within 5 days.
| Original language | English (US) |
|---|---|
| Title of host publication | Chromosomal Mutagenesis |
| Subtitle of host publication | Second Edition |
| Publisher | Springer Fachmedien |
| Pages | 133-159 |
| Number of pages | 27 |
| ISBN (Electronic) | 9781493918621 |
| ISBN (Print) | 9781493918614 |
| DOIs | |
| State | Published - Nov 19 2014 |
Keywords
- Genome engineering
- Golden Gate assembly
- Site-specific nuclease
- TALEN
- Targeted mutagenesis